1. Field of the Invention
The present invention is directed to nucleic acid and host cells useful in controlling the production of polypeptides in bacterial host cell cultures. More particularly, the invention relates to nucleic acid encoding PstS variants having mutations in the phosphate-binding region of the native PstS protein that allow regulation of induction of polypeptide synthesis in bacterial cells.
2. Description of Related Art
The pstS (phoS) gene encodes a phosphate-binding periplasmic protein that is part of the high-affinity phosphate transport system mediating phosphate uptake in certain prokaryotic organisms such as E. coli with a dissociation constant for phosphate of less than 1 .mu.M. Medveczky and Rosenberg, Biochim. Biophys. Acta, 211: 158-168 (1970). The molecular structure of the phosphate-transport protein is provided in Luecke and Quiocho, Nature, 347: 402-406 (1990).
The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by, the regulatory regions of the genes in the pho or pst regulon. Over twenty genes are regulated by phosphate, including pstA, pstS, phoE, pstB, phoU, and ugpAB. When the phosphate concentration of the media drops below about from 0.1 .mu.M to 0.2 mM (Torriani, Biochim. Biophys, Acta, 38: 460-469 [1960]), or in a pstS- mutant (Amemura et al., infra), expression of these genes is induced by a regulatory system that requires the positive regulators PhoB and PhoR.
For an overview of the phosphate regulon in E. coli, see Shinagawa et al., "Structure and Function of the Regulatory Genes for the Phosphate Regulon in Escherichia coli" in Phosphate Metabolism and Cellular Regulation in Microorganisms, Torriani-Gorini et al., eds. (American Society for Microbiology, Washington, D.C., 1987), pp. 20-25; Wanner, "Phosphate Regulation of Gene Expression in Escherichia coli," in Neidhardt FC et al. (eds.) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (American Society for Microbiology, Washington, D.C., 1987) p. 1326-1333; Torriani, BioEssays, 12: 371-376 (1990) ; Matin et al., Annu. Rev. Microbiol., 43: 293-316 (1989).
The DNA fragment containing the pstS gene has been isolated from E. coli strain K-12 chromosomal DNA. Iwakura et al., J. Biochem., 92: 615-622 (1982). Later, the complete nucleotide sequence of, and amino acid sequence encoded by, the pstS gene and prepstS gene were reported by Magota et al., J. Bacterial., 157: 909-917 (1984). See also Surin et al., supra. The pre-PstS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PstS protein, which has the general characteristics of a signal peptide. The mature PstS protein is composed of 321 amino acids with a calculated molecular weight of about 34,422-34,427. The regulatory region of the pstS gene contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence. The sequences of the structural pstS gene and promoter region are also described by Surin et al., Bacterial., 157: 772-778 (1984), who identify an alternative promoter region on the basis of homology with the promoter regions of the pstA and pstE genes. The promoter of the pstS gene was also studied by Kimura et al., Mol, Gen. Genet. 215: 374-380 (1989).
The function of the PstS protein is to transport inorganic phosphate from the periplasm into the cell, as a phosphate-specific transport protein. The transport is achieved when the PstS protein binds to the phosphate through its phosphate-binding domain. For E. coli, this domain includes the backbone residues 10, 11, 38, 140, and 141 and the side chains of residues 10, 38, 56, 135, 139, and 141. Other residues may also indirectly affect phosphate binding, the associated conformational shift from open to closed complex when phosphate is bound to PstS, and/or the associated signaling pathway.
All defined pstS mutations in the PST region were found to lack the periplasmic phosphate-binding protein, so this locus was considered as the structural gene of the binding protein. Levitz et al., Mol, Gen, Genet., 200: 118-122 (1985).
The alkaline phosphatase (phoA) promoter has been used often as a promoter for expressing both homologous and heterologous DNA in bacterial cells. See, e.g., JP 61/280292 published 10 Dec. 1986. In the production of polypeptides utilizing the alkaline phosphatase or pstS promoter cell growth occurs initially with low inorganic phosphate in the medium. These cells utilize the phosphate in the medium so that induction of expression of the gene encoding the polypeptide occurs in late log phase of cell growth as the phosphate content decreases below a threshold value. The cells then starve completely for phosphate, resulting in cessation of growth, a several-fold increase in degradation of cell proteins, so and an inhibition of RNA synthesis. St. John and Goldberg, J. Bacteriol., 143: 1223-1233 (1980). In addition, the extent of expression and rate of protein production cannot be controlled because of the necessity for the near absence of inorganic phosphate in the medium.
Various methods have been explored using the pst regulon to increase expression levels. For example, an expression vector containing a gene coding for PstS bound to a replicon is reported to increase expression levels in bacteria of genes of interest. U.S. Pat. No. 4,703,005 issued 27 Oct. 1987. Additionally, a fusion polypeptide of the sequence PstS-Sc-X-, wherein Sc is a sequence coding for a cleavage site and X is the gene coding for a specified protein, is disclosed in Fr. Pat. Appln. No. 2,599,380 published 4 Dec. 1987.
Mutants of phosphate-specific transport proteins have also been reported. For example, E. coli strains have been described that contain pstA mutants prepared by mixing the bacteria with N-nitroso compounds. Israeli Pat. Appl. No. 60714/3 dated 31 Jul. 1980. Also, strains of E. coli have been reported that specifically excrete alkaline phosphatase, have a mutation in the pst regulon (including a pstS-type mutation) and are transformed by a plasmid containing an E. coli DNA fragment corresponding to the 8.5-minute region of the genetic map. WO 86/04089 published 17 Jul. 1986. E. coli PhoA mutants prepared in such strains have also been described. IL 60,714 published 31 Jul. 1980. Mutated alkaline phosphatase enzymes produced by E. coli with at least one amino acid mutation having increased enzymatic activity over the wild-type enzyme have been disclosed. EP 441,252 published 14 Aug. 1991.
In addition, the PstS function was examined by analysis of 12 pstS mutants, eight of which had a change of Thr-10 to Ile-10, two of which had a change of Ser-254 to Phe-254, one of which had two changes of Thr-10 to Ile-10 and Gly-140 to Glu-140, and one of which had three changes of Thr-10 to Ile-10, Thr-253 to Ile-253, and Ser-254 to Phe-254. The authors postulated from the results that Thr-10 and Ser-254 are involved in the interaction with the membrane components of the Pst system, whereas Gly-140 is involved in the binding of phosphate, or alternatively, there may be more than one phosphate-binding domain in the phosphate-binding protein, and Thr-10 or Ser-254 may also be involved in phosphate binding. Nakata et al., "Genetic and Biochemical Analysis of the Phosphate-Specific Transport System in Escherichia coli," in Phosphate Metabolism and Cellular Regulation in Microorganisms, Torriani-Gorini et al., eds., supra, pp. 150-155.
It is an object of the present invention to identify novel nucleic acid molecules encoding specific variants of PstS that, when integrated into the chromosome of bacterial cells as a replacement for the wild-type pstS gene, will allow growth of bacterial cells transformed with DNA encoding a polypeptide of interest under the control of the alkaline phosphatase promoter in the presence of inorganic phosphate at all growth phases.
It is another object to utilize the novel nucleic acid molecules herein to control the transcription rate of nucleic acid encoding a polypeptide of interest and therefore control the extent is of induction of the alkaline phosphatase promoter in bacterial cells.
It is yet another object to minimize proteolysis of polypeptides produced by bacterial cells under transcriptional control of the alkaline phosphatase promoter.
It is still another object to control the strength of induction of the alkaline phosphatase promoter to minimize cell toxicity caused by rapid induction of the promoter.
These and other objects of the invention will be apparent to the ordinary artisan upon consideration of the specification as a whole.